US20090214595A1

US20090214595A1 - Means for Obtaining Avirulent Leishmania Promastigotes, Promastigotes Obtained, and the Applications Thereof

Info

Publication number: US20090214595A1
Application number: US11/884,015
Authority: US
Inventors: Jean-Loup Lemesre; Philippe Holzmuller; Rachel Bras-Goncalves
Original assignee: Institut de Recherche pour le Developpement IRD
Current assignee: Institut de Recherche pour le Developpement IRD
Priority date: 2005-02-10
Filing date: 2006-02-10
Publication date: 2009-08-27
Also published as: FR2881752A1; EP1851316B1; CA2597512A1; FR2881752B1; WO2006085011A1; ZA200706966B; BRPI0607548A2; AU2006212121A1; US8187870B2; CN101155919A; EP1851316A1

Abstract

The invention relates to an expression vector of a gene coding for an antigenic protein of Leishmania promastigote, characterised in that it comprises a PSA gene insert in opposite orientation. The invention can be applied to the development of mutants under-expressing, or no longer expressing, genes coding for an antigenic protein of Leishmania promastigote, and to the therapeutic and/or vaccine-oriented uses thereof.

Description

The invention relates to means for obtaining avirulent leishmania promastigotes. It also relates to the promastigotes obtained and to the uses thereof, in particular in immunoprophylaxis.
Leishmaniases constitute worldwide endemic parasitic infections which represent a considerable public health problem affecting both developing countries and countries such as those of southern Europe, where they are considered to be opportunistic diseases in immuno-depressed individuals.
At the current time, no effective immunoprophylactic or immunotherapeutic means exist against leishmaniases, and more particularly against the responsible infectious agent, namely leishmania parasites.
Leishmania parasites are flagellate protozoa belonging to the Trypanosomatidae family and to the Leishmania genus. This family includes parasites which can infect humans, and certain animals such as rodents, canines, or mammals.
The biological cycle of protozoa of the Leishmania genus is shared between an invertebrate vector host, the sandfly (small hematophagous dipterous insect), and many vertebrates, including humans. The leishmania have a heteroxenous parasitic developmental cycle during which two distinct morphological stages follow on from one another. In the sandfly, the parasite is in a mobile, extracellular, flagellate form inside the digestive tube of the insect, called promastigote stage. In the invertebrate host, the parasite transforms into an immobile ovoid form of from 2 to 6 μm in diameter, with a very short flagellar residue, called amastigote, which will reside in the parasitophorous vacuole, in the cells of the reticulohistiocytic system, mainly macrophages.
The female hematophagous vector insect becomes infected via the blood of the contaminated vertebrate host, by ingestion of parasitized macrophages. Most of the ingested amastigotes are then destroyed in the following hours. The survivors transform into promastigotes, which will colonize the median intestine, where they actively multiply. They subsequently migrate to the anterior and buccal parts of the digestive tract and then undergo differentiation leading to the formation of metacyclic promastigotes which are highly infectious for the vertebrate host.
The vertebrate host is contaminated with the metacyclic promastigotes when it is bitten by an infected female sandfly. These promastigote forms are injected into the dermis together with saliva, and will then be phagocytized by the cells of the reticulohystiocytic system, in particular macrophages, inside which they differentiate into immobile intracellular amastigote forms. They will then actively multiply by schizogenesis inside the parasitophorous vacuole (phagolysosome) until lysis of the host cell is brought about, thus allowing the released amastigotes to propagate the infection by colonizing other normal macrophages. A further bite by a sandfly will result in infection of the latter and will allow the parasite to perpetuate its cycle.
Despite the advances in this field, leishmaniases still remain today a serious public health problem due to the number of individuals involved, the extent of the geographic area covered by the disease, and the diversity and seriousness of the clinical pictures observed.
The need for effective vaccines against leishmaniases and for novel therapeutic and/or vaccine-related targets for treating these parasitic diseases can therefore be seen.
During infection, the infectious promastigote forms injected into the mammalian host must evade the host's immune response in order to be able to subsequently invade the macrophages. The parasite must therefore implement mechanisms which allow it to escape this immune response and to be capable of adapting to a new environment. Early recognition of the parasite by the host organism during invasion may thus be a key step in resistance to the infection.
Surface antigens, lipophosphoglycan (LPG) and gp63 (metalloprotease), also called leishmanolysin, were discovered in the 1980s.
LPG is the major surface molecule of the promastigote forms, the role of which as a virulence factor has been confirmed.
gp63 is a major surface protease, also considered to be a virulence factor.
Antigens of a third type, called PSAs for “Promastigote Surface Antigens”, were discovered in 1988-1989. They constitute the major surface antigens of the promastigote forms.
Excretory/secretory antigens (ESAs), which have been very widely studied in many parasitic microorganisms (nematodes, protozoa, etc.), play an important role in the infection and defense mechanisms of these parasites, since they have the possibility of acting remotely. In leishmania, research studies have for a long time been limited by the difficulty in being able to have ESAs available in sufficient amounts to study them.
All these antigens appear to play a determining role in the survival and infectiousness of the promastigote forms.
Prior studies by the inventor in this field had led to the development of completely defined culture media, without macromolecules and free of fetal calf serum, allowing the continuous culture of the promastigote and amastigote forms under axenic and serum-free conditions. These media, described in particular in patent application FR No. 93 05 779, have made it possible to reproduce, in vitro, the complete parasitic developmental cycle of leishmania, such as L. amazonensis and L. infantum, and to have, under natural conditions, a relatively abundant source of ESAs available by simply concentrating the culture medium supernatant metabolized by the parasites. A characterization of leishmania ESAs could thus be carried out.
Studies performed in the inventors, laboratory have thus shown that the major immunogen of the L. amazonensis ESAs has a molecular mass of 45 kDa. The screening of a promastigote-form cDNA expression library with a monoclonal antibody called E2, produced against this major immunogen, has made it possible to isolate and identify cDNA clones encoding proteins of the PSA family. This antibody is produced by the murine hybridoma IV D6/E2 deposited with the C.N.C.M, 28 rue du Dr Roux, Paris, France, on Nov. 10, 2004, under the No. I-3318, more especially by the murine hybridoma deposited on Feb. 3, 2006, with the CNCM under the No. I-3570. Thus, for the first time, three new PSA cDNA sequences expressed in the promastigote form of L. amazonensis were identified. These isolated cDNAs differ from one another by virtue of the number of LRRs (4, 6 or 7). More recently, a screening of an L. infantum promastigote cosmid library has been carried out using, as radiolabeled probe, the DNA encoding L. amazonensis PSA. A new Leishmania gene, IJ11, has been identified, encoding an excreted/secreted protein of L. infantum, of molecular mass 56 kDa (LiPSA 50s), belonging to the PSA family.
A functional study of the genes encoding PSAs, major immunogens of the excretory/secretory products of leishmania, was therefore undertaken using the tools provided by genomics. The experimental approach consisted in introducing, into the promastigotes of a leishmania, plasmid constructs capable of inducing a decrease in the level of production of the PSA in order to study the repercussions of these changes on the biology of the parasite and its virulence.
It thus appeared that the nondirectional cloning, in an expression vector for the IJ11 gene encoding the LiPSA 50s protein, resulted, after selection, in plasmids being obtained which contain the insert in an antisense orientation (Ldi antisense), allowing the under-expression of the PSA in the modified L. infantum promastigotes.
Advantageously, it was found that these results could be generalized both as regards the leishmania species and as regards the gene inserts encoding antigenic proteins that can be used.
The aim of the invention is therefore to provide novel expression vector constructs for genes encoding antigenic leishmania promastigote proteins.
The invention is also directed toward the use of these constructs for obtaining avirulent leishmania promastigote mutants.
According to yet another aspect, the invention is directed toward the uses of these avirulent mutants for immunoprophylactic purposes.
The expression vectors according to the invention are characterized in that they contain an insert, in an antisense orientation, of a gene encoding an antigenic leishmania promastigote peptide or protein.
Advantageously, the cloning into expression vectors of said genes in an antisense orientation allows the peptide or the protein to be underexpressed in the modified leishmania promastigotes.
Preferably, said vectors are characterized in that they contain a leishmania PSA gene insert.
In one embodiment of the invention, it is an L. amazonensis gene insert.
The L. amazonensis PSA genes are more especially chosen from the group comprising SEQ ID Nos. 1 to 5.
In another preferred embodiment of the invention, it is an L. infantum gene insert.
The L. infantum genes are advantageously chosen from the group comprising SEQ ID No. 6.
The invention is also directed toward genetically modified leishmania promastigotes, characterized in that they are avirulent mutants, transfected with a vector as defined above.
The invention is also directed toward a method of obtaining avirulent forms of leishmania promastigotes, characterized in that it comprises:

- nondirectional insertion, into an expression vector, of genes encoding antigenic leishmania peptides or proteins,
- selection of the constructs containing the inserts in an antisense orientation,
- transfection of leishmania promastigote forms using the selected constructs,
- biological cloning of the genetically transformed promastigotes by micromanipulation so as to obtain mutants which underexpress, or even which no longer express, the gene encoding an antigenic leishmania promastigote protein.

The transfection of the promastigote forms is advantageously carried out by electroporation.
Revelation of the peptide or of the protein by Western blotting makes it possible to estimate its level of production in the clones.
The use of the above arrangements makes it possible to study the consequences of the underexpression of antigenic leishmania peptides or proteins on the behavior of the transformed promastigotes and to thus identify their biological role.
To this effect, the various genetically modified parasites were cloned by micromanipulation (biological cloning) and characterized and compared at the molecular level (level of production of the antigenic protein, level of expression of the corresponding transcripts). The phenotypic characterization of the genetically modified and cloned promastigotes consisted in comparing the growth kinetics of these parasites and also their infectious capacity in vitro with respect to canine macrophages.
The characterization of a parasitic virulence factor, such as PSA, makes it possible to develop novel strategies for interference with the infectious process for therapeutic and/or vaccine-related purposes.
In particular, the invention makes it possible to obtain attenuated, or even avirulent, strains that can be used in animal and/or human vaccination.

Other characteristics and advantages of the invention are given in the examples which follow with reference to FIGS. 1 to 4, which represent, respectively,

FIGS. 1 and 2, the results of analysis by Western blotting of the PSA of total polypeptide extracts of a wild-type strain of Ldi Mon1 and of excretory/secretory antigens of culture supernatants of wild-type strains, of sense and anti-sense transformed parasitic parent strains and of clones thereof;

FIG. 3, results of RT-PCR on transcripts of a gene encoding PSA and of a housekeeping gene;

FIG. 4, the parasitic index determined over the course of the in vitro infection of canine macro-phages with a wild-type strain or clones.

MATERIALS AND METHODS

I—In vitro Cultures

1—Axenic Cultures of L. infantum
The wild-type strain of Leishmania infantum used in the studies described hereinafter bears the WHO reference: MHOM/MA/67/ITMAP-263/clone2, zymodeme MON-1. The transformed strains were obtained after cloning of the IJ11 gene (1404 bp), encoding the L. infantum PSA (56 kDa), in the sense or antisense position in the expression vector pTEX, and then transfection by electroporation into the promastigote form of L. infantum. The Ldi IJ11 antisense, Ldi pTEX transformed parasites were selected in the presence of increasing concentrations of geneticin up to 50 μg/ml. The presence of the plasmid and the orientation of the transgene (antisense) were verified by Southern blotting and the level of transcription of the transgene was verified by Northern blotting.
The promastigote forms are cultured under axenic conditions in a completely defined culture medium containing no macromolecule as serum substitute (serum-free RPMI 199H medium, pH 7), but also in RPMI 1640 medium containing 20% of decomplemented fetal calf serum (20% serum RPMI medium, pH 7.2), and supplemented with penicillin (100 units/ml) and with streptomycin (100 μg/ml). The various transformed strains were cloned by the micromanipulation technique. A single parasite is sampled under a microscope using a fine glass needle and deposited on NNN medium (Novy, macNeal, Nicolle). The culturing of the parasites is carried out by successive subculturings in 20% RPMI medium. The genetically modified promastigote forms and the corresponding clones are maintained in continuous culture by weekly subculturings in serum-containing culture media or adapted in serum-free medium at 25° C. in the presence of geneticin (50 μg/ml). The parasite concentration is determined by flow cytometry (FacsCalibur, Beckton Dickinson). The parasites are conserved by cryofreezing of the organisms in culture in the presence of 3% DMSO (dimethyl sulfoxide) at −180° C. in liquid nitrogen.
2—Macrophage Cultures
PBMCs (Peripheral Blood Mononuclear Cells) are isolated from whole blood of a normal dog by Ficoll gradient (Ficoll Lymphoprep®, density 1.077±0.001 g/ml; AbCys, France) and by centrifugation at 700 g for 20 min at ambient temperature. The cells are then washed 3 times in PBS (0.01M phosphate buffered saline, pH 7.2) without Ca²⁺/Mg²⁺by centrifugation at 400 g for 10 min at 4° C., before being resuspended in RPMI 1640 supplemented with 10% of fetal calf serum, 100 units/ml of penicillin and 100 μg/ml of streptomycin, and then cultured at 37° C.

II—Harvesting and Preparation of the Parasite Material

1—Harvesting of the Parasite Material
The promastigote forms in the stationary growth phase (7 days) are harvested by centrifugation of the serum-free parasite cultures at 3000 g for 10 min at 4° C. The parasite pellets and the culture supernatants thus dissociated are then treated separately.
2—Preparation of Parasite Lysates
The parasite pellet is washed twice in PBS under the centrifugation conditions described above. After the final wash, the number of parasites harvested is determined by flow cytometry. 10⁹washed parasites are resuspended in a hypotonic lysis solution: 50 mM Tris, pH 8, containing 1% of Triton X100 (Sigma). After incubation at 4° C. for 20 min, centrifugation for 20 min at 4° C. and at 2000 g makes it possible to separate the insoluble material (debris) from the supernatant (soluble molecules). This supernatant corresponds to a total polypeptide extract (TPE).
3—Concentration of the Excretory/Secretory Antigens (ESAs) from the Culture Supernatant Metabolized by the Parasites
10 ml of supernatant metabolized by the parasites are filtered through a Millipore membrane (0.22 μm) and then frozen in shells before being freeze-dried. The freeze-dried materials are taken up in 1 ml of milliQ water in order to be dialyzed for 48 h at 4° C. so as to remove the salts. A second freeze-drying process makes it possible to concentrate the supernatant 200-fold.

III—Biochemical Analyses

1—Protein Assay
The assaying of the proteins is carried out using the Beckman DU-600 spectrophotometer, at 595 nm, according to the Bradford method, using the Biorad reagent (Biorad Protein Assay Kit 11). A standard range, from 5 to 20 μg/ml, is prepared from a standard bovine albumin solution (1 mg/ml BSA, Sigma).
2—SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) Analysis
The SDS-PAGE polyacrylamide gel analysis was carried out in a mini-gel electrophoresis tank (Biorad). The acrylamide percentages used are 5% for the stacking gel and 10% for the separating gel, thereby making it possible to separate proteins of 10 to 200 kDa. TPE protein concentrations are mixed (v/v) with sample buffer containing 4% SDS, 20% glycerol and 0.5% bromo-phenol blue in 0.5M Tris-HCl, pH 6.8.
The electrophoretic migration is carried out in a buffer (2.5 mM Tris/19.2 mM glycine, pH 8.3) containing 0.1% SDS under a constant voltage of 80V. The calibration of the gel is obtained by depositing 10 μg of standard proteins of known molecular weights (Mark12 Wide Range Protein Standard/SeeBlue® Plus2 Pre-Stained Standard, Novex).
3—Revelation of the Proteins with Silver Nitrate
After removal of the SDS by washing in the presence of acetic acid (10%) and ethanol (40%) for 1 h, the acetic acid is then itself removed by two rinses for 30 min in 50% ethanol. The gels are then incubated for 15 min in a staining mixture: 19.4% AgNO₃-0.36% NaOH. After 5 washes in milliQ water, a revealing solution containing 0.02% formaldehyde in 1% citric acid (w/v) reveals the proteins on the gel. The revelation threshold for this staining method is 2 ng of protein equivalent.

IV—Immunoanalyses

1—Western Blotting
After having been separated in an SDS-PAGE polyacrylamide gel, the proteins are blotted onto a nitrocellulose membrane (Amersham, Hybond-C Extra). The blotting is carried out under semi-dry conditions. The membranes are then saturated for one hour in a TBS-5% milk blocking solution. The membranes are incubated overnight at ambient temperature and with agitation, with the F5 monoclonal antibody directed against PSA and obtained in mice (MAB F5), diluted to 1/50 in TBS. After 3 washes for 5 min in TBS, the bound antibodies are detected with peroxidase-coupled anti-mouse immuno-globulin G secondary antibodies (Tebu) diluted to 1/500 in TBS and incubated for 45 min at ambient temperature with agitation. Next, 3 washes for 5 min in TBS are carried out before revealing the reaction with a solution containing 15 mg of 4-chloro-1-naphthol dissolved in 5 ml of methanol, to which are added 25 ml of TBS containing 15 μl of 30% H₂O₂. The reaction is stopped by washing the membrane with distilled water.
2—Indirect Immunofluorescence on Parasites
The parasites in the stationary phase of their culture (7 days) are fixed with 1% formaldehyde in PBS without Ca²⁺/Mg²⁺, and then washed. Once fixed, the parasites are deposited on Teflon-coated wells (10 wells, 50 ml/well) and dried in the open air. Each well is then processed with the F5 monoclonal antibody (MAB F5) diluted in a (saturating) blocking solution (1% goat serum, 0.5% BSA in PBS) for 30 min at 37° C. The cells are then washed and incubated for 30 min at 37° C. with the second antibody conjugated to fluorescein isothio-cyanate (FITC) (goat anti-mouse immunoglobulin G anti-serum) diluted to 1/300 in a solution of PBS containing Evans blue at 1/7500. The slides are observed under a photon fluorescence microscope (Leitz).

V—Molecular Analysis

1—Total RNA Extraction
The promastigote forms are harvested by centrifugation of the serum-free parasite cultures at 3000 g for 10 min at 4° C. The parasite pellet is washed twice in PBS under the above centrifugation conditions. After the final wash, in order to lyse the parasites and to inhibit the endogenous ribonucleases (RNAses), the cells are dissociated by vortexing in 1 ml of Trizol (Trizol Reagent, GibcoBRL), and then incubated for 5 min at ambient temperature. The lysate is incubated in 0.2 ml of chloroform for 2 min at ambient temperature, and then centrifuged at 12 000 g for 15 min at 4° C. so as to give two phases, an aqueous phase and an organic phase. The RNAs are exclusively in the aqueous phase, which is recovered. The RNAs are then precipitated by adding 0.5 ml of isopropanol and incubated for 10 min at ambient temperature, and then centrifuged at 12 000 g for 10 min at 4° C. The pellet is washed in 1 ml of 75% ethanol, and then centrifuged at 7500 g for 5 min at 4° C., before being taken up in 40 μl of water (RNAse-free). In order to remove any possible presence of DNA, the dissolved RNA pellet is taken up in 1 μl of DNAse I+0.1 volume of 10× DNAse I buffer, and incubated for 20 min at 37° C. After the addition of 5 μl of DNAse inactivation reagent, incubation for 2 min at ambient temperature and then centrifugation at 10 000 g for 1 min at 4° C., the supernatant containing the RNAs is recovered. The total RNA is quantified by spectrophotometry, the absorbance being read at 260 nm (1 OD unit=40 μg/ml of single-stranded RNA). The quality of the extracted RNA and the amount and the size are verified by electrophoresis on 1% agarose gel in 0.5× TAE buffer, in the presence of 0.01% ethidium bromide (ETB). The size marker used is the SmartLadder (Eurogentec). The total RNA is then visualized under UV at 312 nm after migration. The selected criteria for quality of the extraction are the absence of DNA and the visualization of 3 ribosomal RNA bands of equal intensity.
2—RT-PCR
The single-stranded cDNA is synthesized by carrying out an RT-PCR with 1 μg of total RNA, 2 μl of dNTP mix (5 mM), 0.2 μl of specific primers (10 μM) and H₂O qs 13 μl. The mix is incubated for 2 min at 65° C. and then for 2 min in ice, before the following are added: 4 μl of 5× buffer, 1 μl of DTT (0.1M), 1 μl of Superase In (40U) (RNAse Inhibitor, Ambion) and 1 μl of SuperScript™ (200U) (Reverse Transcriptase, Invitrogen). The mix is incubated for 50 min at 42° C., and the reaction is then inactivated by heat shock for 15 min at 70° C.
The single-stranded cDNA is amplified by PCR with 3 μl of cDNA, 12.5 μl of PCR Mastermix (Promega), 1 μl of 5′P and 3′P specific primers (10 μM) and H₂O qs 25 μl. The following primers were used:

SEQ ID No. 7	-nhinsp1:	5′-CATCGCTTACCCCTTGTT-3′

SEQ ID No. 8	-nhinsp2:	5′-GTGGCCAATCGCGACA-3′

SEQ ID No. 9	-P1RT:	5′-ATGGCGCTGTGCGTGCGTCGG-3′

SEQ ID No. 10	-P4RT:	5′-GCGCGCGACAGCAGCGGCAT3′.

The program of the PCR reaction used and carried out in a thermocycler (GeneAmp PCR System 2400, Applied Biosystems) is the following: 5 min at 94° C., then 27 cycles (94° C. 30 sec, 55° C. 1 min, 72° C. 1 min) and 10 min at 72° C.
The PCR products are verified by 1% agarose gel electrophoresis.

VI—Biological Analysis

1—Growth Kinetics
The in vitro culture kinetics were realized over 10 days in RPMI 20% medium, starting from an initial concentration of 5×10⁵parasites/ml. The parasite concentration is determined by flow cytometry (FacsCalibur, Beckton Dickinson), by mixing 500 μl of PBS, 5 μl of culture and 0.5 μl of propidium iodide.
2—In vitro Infection in Parasite/Macrophage Interactions
10⁶PBMCs/well (200 μl), isolated from normal dog whole blood, are distributed in LabTek® culture chambers (Nalge Nunc International). After incubation for 30 min at 37° C., 5% CO₂, the LabTek® are washed gently with RPMI 10% in order to remove the cells which have not adhered. The macrophages are obtained from the cultured monocytes after 5 days of maturation. The mature macrophages are infected in vitro with promastigote forms in the stationary culture phase, for 30 min, 2 h and 48 h at 37° C. and 5% CO₂at a parasite:macrophage ratio of 10:1. The macrophages are then washed gently twice in order to remove the nonphagocytozed parasites. After 30 min, 2 h and 48 h, the cells are fixed with methanol and stained with Giemsa. 300 macrophages are observed under an immersion optical microscope (1000× magnification), and the percentage of parasitized macrophages, the number of amastigotes per macrophage and the parasitic index (P.I.) are determined. The P.I. is equivalent to the percentage of infected macrophages multiplied by the number of amastigotes per macrophage.

RESULTS

Evaluation of the Level of Production of PSA in the Transformed Clones
FIGS. 1 and 2 report the results of Western blotting analysis of the PSA, respectively,

- of total polypeptide extracts (TPE) derived from the parasite pellets of the wild-type strain Ldi Mon1,
- on excretory/secretory antigens (ESAs) derived from metabolized culture medium supernatants of the wild-type strain Ldi Mon1, of the parent strains of transformed parasites (Ldi pTEX, Ldi IJ11 sense and Ldi IJ11 antisense) and of their corresponding clones (5 μg protein equivalent of total extract per lane). It is noted that no band is observed for the wild-type strain Ldi Mon1, that the overexpression is, on the other hand, clearly visible for Ldi IJ11 sense, both at the level of the TPEs and the ESAs, compared with the expression in Ldi pTEX, and that a decrease in the expression of the PSA is observed with Ldi IJ11 antisense.

Analysis of the PSA Transcripts by RT-PCR
An RT-PCR was carried out on the transcripts of the gene encoding the PSA and also on the transcripts of a housekeeping gene encoding nucleoside hydrolase.
The results obtained are given in FIG. 3. A significant increase in the level of the transcripts encoding the PSA is observed in Ldi IJ11 sense, and a notable decrease is observed in Ldi IJ11 antisense, compared with Ldi pTEX. No signal is observed with the wild-type strain of Ldi Mon1.
In vitro Infectious Capacity in the Parasite-Macrophage Interactions
FIG. 4 reports the parasitic index determined during the in vitro infection of canine macrophages with the wild-type strain or the various selected clones at various incubation times: 30 min (parasite attachment to the macrophages), 2 h (parasite penetration) and 48 h (survival and multiplication of amastigotes).
It is noted that, at 30 min and at 2 h, the Ldi IJ11 sense promastigotes exhibit a parasitic index which is approximately two times higher than those of Ldi IJ11 antisense is approximately two times lower. The same results are obtained after 48 h of infection.

VII—Study of the In vivo Role of the pTEX, Sense and Antisense Mutant

Infection of Balb/C Mice by IP Injection with 10⁸Promastigotes/100 μl
Protocol for Measuring Parasitemia (Parasite Burdens)

- Bibliographic reference: P. A. Buffet et al., 1995. Antimicrobial agents and chemotherapy. Vol. 39: 2167-2168
- Prepare: 1 tube of 15 ml per organ with 4 ml of SDM or RPMI medium/20% FCS Weigh the tubes+medium (weight=T in g) Cell Strainer+small Petri dishes (1 cell strainer for two same organs belonging to the same group/batch of mice)+1 ml syringe

On spleen and liver organs:
For the spleen: remove the entire spleen
For the liver: remove only the small lobe

- Weighing of organs (organ in tube+medium, weight=TO in g)
- Grinding/homogenization with a Potter homogenizer or cell strainer in 4 ml of medium.

For parasitemia/spleen:

- In a 96-well plate, remove 250 μl of cells for the 1st well and prepare doubling dilutions with 150 μl of parasites in 150 μl of medium, and then, for the following wells, remove 150 μl to be diluted in 150 μl of medium, etc. Prepare 2×12 wells of dilutions (2 rows).
- After incubation for 7 and 15 days at 26-28° C., the plates are examined under an inverted microscope (×100 or ×200 objective).
- Note the absence or the presence of parasites for each well. Note the last well where parasites are present.

For parasitemia/liver:

- In a 96-well plate, remove 25 μl of cells for the 1st well containing 225 μl of medium and prepare 4-fold serial dilutions with 50 μl of parasites in 150 μl of medium, and then, for the following wells, remove 50 μl to be diluted in 150 μl of medium, etc. Prepare 2×12 wells of dilutions (2 rows).
- After incubation for 7 and 15 days at 26-28° C., the plates are examined under an inverted microscope (×100 or ×200 objective).
- Note the absence or the presence of parasites for each well. Note the last well where parasites are present.

The final titer corresponds to the last dilution for which the well contains at least 1 parasite.
The number of parasites per gram (parasite burden) in the corresponding organ is calculated in the following way:
Number of parasites=(geometric mean of the inverse titers of each duplicate/weight of section homogenized)×400
$Parasite burden = \frac{inverse of the limiting dilution}{organ weight (g)} \times 400$
N.B.:—400 corresponds to the inverse fraction of the homogenized organ inoculated in the 1st well

- <10³to >10⁶

Point at 14 days after infection:
Parasitemia (reading at 15 days) carried out in duplicate and doubling dilutions
Parasite burden=(inverse of the limiting dilution/organ weight (g))×400


					General
Organ		Parasite	Mean		mean
weight	Limiting	burden	Parasite	Standard	Parasite	Standard
(g)	dilution	Parasite g-1	burden	deviation	burden	deviation g

Batch

1 pTEX

Mouse

1 spleen	0.14	0.0078	366300	274725	129507	107912	56756	without mouse 1 trial
	0.14	0.0156	183150	183150
Mouse 2 spleen	0.16	0.0156	160256	120064	56840			without mouse 1 trial
	0.16	0.0313	79872			67348	22927	1 and 2 and
Mouse 3 spleen	0.14	0.0625	45714	68498	32222			without mouse 2 trial
	0.14	0.0313	91283
Mouse 4 spleen	0.16	0.0625	40000	59936	28194
	0.16	0.0313	79872

Batch 2 sense

Mouse

1 spleen	0.14	0.5	5714	2857	4041	2367	1517
	0.14	nothing	0
Mouse 2 spleen	0.19	nothing	0	2105	2977
	0.19	0.5	4211
Mouse 3 spleen	0.24	1	1667	833	1179
	0.24	nothing	0
Mouse 4 spleen	0.17	0.25	9412	7059	3328
	0.17	0.5	4706	4706
Mouse 5 spleen	0.15	1	2667	1333	1886
	0.15	nothing	0

Batch 3 antisense

Mouse

1 spleen	0.13	0.25	12308	12308	0	8744	2458
	0.13	0.25	12308
Mouse 2 spleen	0.14	1	2857	1429	2020
	0.14	nothing	0
Mouse 3 spleen	0.15	0.5	5333	8000	3771
	0.15	0.25	10667
Mouse 4 spleen	0.18	0.25	8889	6667	3143
	0.18	0.5	4444
Mouse 5 spleen	0.20	0.25	8000	8000	0
	0.20	0.25	8000

results in red not taken into account


Summary point 15 d

Mean
Parasite	Standard	Mean
burden	deviation	deviation

Batch

1 pTEX

Mouse

1 spleen	274725	129507	91575
	Mouse 2 spleen	120064	56840	40192
	Mouse 3 spleen	68498	32222	22784
	Mouse 4 spleen	59936	28194	19936

Batch 2 sense

Mouse

1 spleen	2857	4041	2857
	Mouse 2 spleen	2105	2977	2105
	Mouse 3 spleen	833	1179	833
	Mouse 4 spleen	7059	3328	2353
	Mouse 5 spleen	1333	1886	1333

Batch 3 antisense

Mouse

1 spleen	12308	0	0
	Mouse 2 spleen	1429	2020	1429
	Mouse 3 spleen	8000	3771	2667
	Mouse 4 spleen	6667	3143	2222
	Mouse 5 spleen	8000	0	0

	Mean
	Parasite	Standard
	burden	deviation

pTEX mouse

2 spleen	120064	56840
	pTEX mouse 3 spleen	68498	32222
	pTEX mouse 4 spleen	59936	28194
	Sense mouse 1 spleen	2857	4041
	Sense mouse 2 spleen	2105	2977
	Sense mouse 3 spleen	833	1179
	Sense mouse 4 spleen	7059	3328
	Sense mouse 5 spleen	1333	1886
	Antisense mouse 1 spleen	12308	0
	Antisense mouse 2 spleen	1429	2020
	Antisense mouse 3 spleen	8000	3771
	Antisense mouse 4 spleen	6667	3143
	Antisense mouse 5 spleen	8000	0

	Parasite	Standard
	burden	deviation

pTEX	82833	3252
Sense	2838	248
Antisense	7281	390


Parasitemia (2 months)

			Parasite		General
	Organ		burden	Mean	mean	Standard
	weight	Limiting	parasite	Parasite	Parasite	deviation
SPLEEN	(g)	dilution	g-1	burden	burden	g

Batch

1 pTEX

Mouse

1 spleen	0.14	0.016	182857	264127	118470
Mouse 2 spleen	0.14	0.008	365714
Mouse 3 spleen	0.17	0.031	75294
Mouse 4 spleen	0.18	0.016	142222
Mouse 5 spleen	0.14	0.008	365714

Batch 2 sense

Mouse

1 spleen	0.1	NI	0	0
Mouse 2 spleen	0.15	NI
Mouse 3 spleen	0.12	NI
Mouse 4 spleen	0.14	NI
Mouse 5 spleen	0.14	NI

Batch 3 antisense

Mouse

1 spleen	0.11 NI		0	1813	2914
Mouse 2 spleen	0.12	0.500	6667
Mouse 3 spleen	0.19	0.500	4211
Mouse 4 spleen	0.13 NI		0
Mouse 5 spleen	0.14 NI		0
Mouse 6 spleen	0.18 NI		0

Parasite burden g-1

Standard deviation

Spleen	15 days	2 months	15 days	2 months

pTEX	107912	264127	56756	118470
Sense	2367	0	1517	0
Antisense	8744	1813	2458	2914
pTEX	67348	264127	22927	118470
Sense	2367	0	1517	0
Antisense	8744	1813	2458	2914

results in red not taken into account
NI = non infected

Parasitemia (2 months)

	Organ		Parasite
	weight	Limiting	burden
LIVER	(g)	dilution	parasite g-1

Batch 1 pTEX				2495238	4025557	with mouse 1
Mouse 1 liver	0.12	0.000391	8533333
Mouse 2 liver	0.1	0.000024	163840000	482540	43989	without mouse 1
Mouse 3 liver	0.14	0.006250	457143
Mouse 4 liver	0.14	0.006250	457143
Mouse 5 liver	0.12	0.006250	533333

Batch 2 sense

Mouse

1 liver	0.17	NI	0	0
Mouse 2 liver	0.18	NI
Mouse 3 liver	0.12	NI
Mouse 4 liver	0.14?	NI
Mouse 5 liver	0.09	NI

Batch 3 antisense

Mouse

1 liver

0.12

NI

0

38889

61162

Mouse 2 liver

0.16

0.025

100000

Mouse 3 liver	0.1	NI		0
Mouse 4 liver	0.08	NI		0

Mouse 5 liver

0.12

0.025

133333

Mouse 6 liver	0.1	NI		0

Claims

1. An expression vector for a gene encoding an antigenic leishmania promastigote protein, characterized in that it comprises an insert of a PSA gene in an antisense orientation.

2. The vector as claimed in claim 1, characterized in that it contains an L. amazonensis gene insert.

3. The vector as claimed in claim 2, characterized in that the L. amazonensis gene corresponds to one of the following sequences: SEQ ID Nos. 1 to 5.

4. The vector as claimed in claim 1, characterized in that it contains an L. infantum gene insert.

5. The vector as claimed in claim 4, characterized in that the L. infantum gene corresponds to SEQ ID No. 6.

6. A genetically modified leishmania promastigote, characterized in that it is an avirulent mutant, transfected with a vector as claimed in claim 1.

7. A method of obtaining avirulent forms of promastigotes, characterized in that it comprises:

nondirectional insertion, into a leishmania expression vector, of genes encoding antigenic leishmania peptides or proteins,

selection of the vectors containing the inserts in an antisense orientation,

transfection of leishmania promastigote forms using the selected vectors,

biological cloning of the genetically modified promastigotes by micromanipulation so as to obtain mutants which underexpress, or even which no longer express, the gene encoding an antigenic leishmania promastigote protein.

8. The use of the promastigotes as claimed in claim 7, for developing vaccines.